There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate and qualitative or quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, dental plaque, gingival crevicular fluid and other biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (sometimes identified as a "ligand") and a compound specifically reactive with that substance (sometimes identified as a "receptor").
Specific microorganisms have been implicated as indicators for a number of periodontal diseases in humans and animals, such as gingivitis and periodontitis. The importance of such diseases is growing in the human population, especially as people live longer, and prevention of such diseases is becoming of considerable importance to dentists, insurance carriers and the health industry in general. In addition, proper dental care for animals is a growing concern in our culture.
Detection of microorganisms associated with periodontal diseases has been accomplished using culture techniques, DNA probes and a number of immunological procedures, such as agglutination assays, enzyme linked immunosorbent assays (ELISA) and others known in the art. ELISA assays utilize the reaction of an extracted antigen from the microorganism(s) and the corresponding antibody to form an immunological complex. Usually uncomplexed materials are washed from the complex in order to provide an accurate assay result.
Extraction of antigen from microorganisms of interest in a biological specimen is generally critical to providing an accurate, rapid and sensitive assay. Many varied techniques have been used for extraction including physical disruption of the cells by sonication, heating or centrifugation. Chemical extraction compositions have also been developed. For example, various surfactants, such as sodium dodecyl sulfate, have been used individually in extraction compositions.
An advance in the art in the detection of microorganisms associated with periodontal diseases is described and claimed in U.S. Ser. No. 468,392 (filed Jan. 22, 1990 by Snyder). This case describes the simultaneous detection and differentiation of these microorganisms, and particularly Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia, in an immunometric (also known as "sandwich") assay using water-insoluble reagents in defined regions of a microporous filtration membrane. The simultaneous detection and differentiation of these microorganisms have considerable clinical and commercial significance.
While the noted simultaneous assay represents an important advance in the art for detecting the noted microorganisms, in some cases, unacceptable background was observed, especially when clinical specimens were tested. It was also noticed that the known surfactant extraction composition did not adequately extract antigen from all serotypes of the microorganisms of interest. This problem was solved by using as an extraction composition, a high pH solution of a cationic surfactant mixed with a specific anionic surfactant. The details of this invention are provided in copending and commonly assigned U.S. Ser. No. 773,833, filed on even date herewith by Snyder, Contestable, Abrams and Grogan and entitled "Test Kit and Method for the Detection of Microorganisms Associated with Periodontal Diseases Using Surfactant Mixture As Extraction Composition" now U.S. Pat. No. 5,334,503.
Further improvements were provided by the use of certain wash solutions in such assays, as described in U.S. Ser. No. 774,019 (filed on even date herewith by Boyer, Contestable and Snyder), entitled "Wash Composition, Test Kit and Method for Determination of Microorganisms Associated with Periodontal Diseases" now U.S. Pat. No. 5,248,595.
However, further improvement is needed since in the assay of some clinical specimens to differentiate among microorganisms, false positives have been observed when one microorganism is present in relatively higher concentrations than the others being detected.